Development 111, 15-22 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
15
T-cadherin expression alternates with migrating neural crest cells in the
trunk of the avian embryo
BARBARA RANSCHT
1
* and MARIANNE BRONNER-FRASER
2
x
La Jolla Cancer Research Foundation, La Jolla, CA 92037, USA
2
Developmental Biology Center, University of California, Irvine, CA 92717, USA
* To
whom correspondence should be addressed
Summary
Trunk neural crest cells and motor axons move in a
segmental fashion through the rostral (anterior) half of
each somitic sclerotome, avoiding the caudal (posterior)
half.
This metameric migration pattern is thought to be
caused by molecular differences between the rostral and
caudal portions of the somite. Here, we describe the
distribution of T-cadherin (truncated-cadherin) during
trunk neural crest cell migration. T-cadherin, a novel
member of the cadherin family of cell adhesion
molecules was selectively expressed in the caudal half
of
each sclerotome at all times examined. T-cadherin
immunostaining appeared graded along the rostrocau-
dal axis, with increasing levels of reactivity in the caudal
halves of progressively more mature (rostral) somites.
The earliest T-cadherin expression was detected in a
small population of cells in the caudal portion of the
somite three segments rostral to last-formed somite. This
initial T-cadherin expression was observed concomitant
with the invasion of the first neural crest cells into the
rostral portion of the same somite in stage 16 embryos.
When neural crest cells were ablated surgically prior to
their emigration from the neural tube, the pattern of
T-cadherin immunoreactivity was unchanged compared
to unoperated embryos, suggesting that the metameric
T-cadherin distribution occurs independent of neural
crest cell signals. This expression pattern is consistent
with the possibility that T-cadherin plays a role in
influencing the pattern of neural crest cell migration and
in maintaining somite polarity.
Key words: trunk neural crest cell migration, cell adhesion
molecules, chick embryos, somite, sclerotome.
Introduction
Neural crest cells initiate their migration from the
neural tube shortly after fusion of the neural folds and
neural tube closure. These cells migrate along several
well-defined pathways and give rise to numerous
derivatives, including pigment cells, Schwann cells,
adrenal chromafnn cells and cartilagenous elements of
the face (ref. LeDouarin, 1982). In addition, neural
crest cells contribute the precursors for most peripheral
neurons and glia. In the trunk region, the neural crest-
derived sensory and sympathetic ganglia with their
associated nerve roots are organized in a segmental
pattern along the vertebral column. This segmental
organization results from the metameric migratory
pattern of neural crest cells that selectively move
through the rostral (anterior) half of each somitic
sclerotome during early phases in development (Rick-
mann
et al.
1985; Bronner-Fraser, 1986; Teillet
et al.
1987;
Lallier and Bronner-Fraser, 1988).
The majority of trunk neural crest cells migrate in a
ventral direction away from the neural tube and
through the adjacent somites. As the epithelial somites
undergo a transition to form the dermatome (presump-
tive dermis), myotome (presumptive muscle), and
sclerotome (presumptive vertebrae), most ventrally
migrating neural crest cells invade the rostral half of
each sclerotome (see Fig. 1); in contrast, the caudal
(posterior) half of each sclerotome lacks migrating
neural crest cells (Rickmann
et al.
1985; Bronner-
Fraser, 1986; Teillet
et
al.
1987; Serbedzija
et
al.
1989).
Similarly, motor axons, emerging from the ventral
neural tube several hours after neural crest cells, extend
their growth cones through the rostral half of the
somitic sclerotome (Keynes and Stern, 1984). For both
populations, the signals responsible for the metameric
pattern appear to reside within the somite
itself.
After
experimental rotation of the segmental plate to reverse
its rostrocaudal polarity, neural crest cells and motor
axons recognize the appropriate rostral portion of the
somite even when presented in a caudal position
(Keynes and Stern, 1984; Bronner-Fraser and Stern,
1991).
Furthermore, in the absence of the somitic
sclerotome, neural crest cells and motor axons migrate
in a non-segmental pattern (Lewis
et
al.
1981; Tosney,
1988;
Stern
et al.
1986; Kalcheim and Teillet, 1989).
16
B. Ranscht and M. Bronner-Fraser
Molecular differences between the cells of the rostral
and caudal halves of the somitic sclerotome are likely to
account for the selective migration pattern of neural
crest cells and motor axons through the rostral half of
the somites. Cells in the rostral half of each somite may
selectively expose or secrete molecules that attract
neural crest cells and/or axons, whereas cells in the
caudal half of each somite may contain molecules that
are repulsive or inhibitory to cell and/or axon
movements. Furthermore, it may be a combination of
both attractant molecules in the rostral and repulsive
molecules in the caudal half of each sclerotome that
regulate the metameric pattern of neural crest cell
and/or motor axon locomotion through somite tissues.
Some molecular differences between rostral and caudal
somites have been noted. Peanut lectin binding
glycoproteins of 48 and
55
x
10
3
M
T
have been localized
specifically to the caudal half of the somitic sclerotome
(Stern
et
al.
1986; Davies
et
al.
1990). In the rostral half
of the sclerotome, a 70xl0
3
M
r
protein (Tanaka
et al.
1989),
butyrylcholinesterase activity (Layer
et
al.
1988),
and cytotactin/tenascin/Jl (Tan
et al.
1987; Mackie
et
al.
1988; Stern
et al.
1989) have been detected. In
addition, differences in the polypeptide composition
between rostral and caudal somite portions have been
revealed by two-dimensional gel electrophoresis (Nor-
ris
et al.
1989).
Cadherins are a class of cell adhesion molecules that
have been suggested to regulate morphogenesis by a
calcium-dependent adhesion mechanism (Takeichi,
1988).
A novel member of this family, T-cadherin
(truncated-cadherin), was recently identified by mol-
ecular cloning and sequencing of corresponding cDNAs
(Ranscht and Dours, 1989; Ranscht and Dours-
Zimmerman, 1991). In the present study, we have
examined the onset and distribution of T-cadherin in
relation to neural crest cell migration. T-cadherin was
localized selectively in the caudal half of the somites at
both initial and advanced stages of neural crest cell
migration. Furthermore, T-cadherin expression in the
caudal half of each somite coincided with the initial
penetration of neural crest cells into the rostral half of
each somite. This distribution is consistent with a
possible role for T-cadherin in influencing the pattern of
trunk neural crest cell migration.
Materials
and
methods
Embryos
White Leghorn chick embryos ranging from stages 11
to 19
(according
to the
criteria
of
Hamburger
and
Hamilton,
1951)
were used
for
this study. Eggs were incubated
in a
forced
air
incubator
at
37 °C until they reached
the
desired stage
of
development.
Fixation
and
tissue processing
Cryostat sections
Embryos were removed from
the
eggs
and
washed
in
Howard's Ringers solution. The embryos were straightened in
wax dishes
by
placing insect pins
or
cactus needles into their
surrounding membranes. They were then fixed
in 4%
paraformaldehyde
in
phosphate-buffered saline (PBS)
at
4
C
C
overnight, washed
in
PBS, transferred
to
5 %
sucrose
in
PBS
with azide
for
4-24 h,
and
immersed
in
15% sucrose
in
PBS
with azide overnight
at 4°C.
Embryos were placed
in 7%
gelatin (Sigma;
300
Bloom)
in
15 %
sucrose/PBS
for
3
h
at
37°C,
and
embedded
in
fresh gelatin. Storage
of
the embryos
was
up to two
weeks
in the
refrigerator until
the
time
of
sectioning when they were rapidly frozen
in
liquid nitrogen.
14
/an sections were cut
on a
Reichardt cryostat
and
mounted
on gelatin-subbed slides.
Paraffin sections
Embryos were fixed
in
Zenkers fixative
for
1.25 h, rinsed
in
running water
for
15min,
and
placed
in 70%
ethanol.
Embryos were dehydrated through
a
series
of
alcohols,
followed
by
three changes
of
histosol,
and
three changes
of
paraffin before embedding in fresh paraffin. Sections were
cut
on
a
Leitz microtome
at a
thickness
of 5/an.
Immunocytochemistry
Cryostat sections were air dried
for
15
min prior to application
of antibody.
A
polyclonal antiserum against T-cadherin
(Ranscht and Dours-Zimmermann, 1991) was applied diluted
1:150 in PBS containing 0.1
%
BSA
and
0.05
%
Triton X-100
at room temperature
for two
hours
or
overnight. Sections
were washed
in
phosphate buffer
and
incubated
for
one hour
with FITC-goat anti-rabbit IgG (Zymed) diluted 1:30
in
0.1
M
PBS,
pH7.4, with
1 %
BSA (PBS/BSA). Slides treated with
preimmune instead
of
immune antiserum gave
no
immuno-
reactivity.
In
some experiments, sections were labelled
simultaneously with anti-T-cadherin and the HNK-1 antibody
which recognizes migrating neural crest cells (Tucker
et al.
1984).
The HNK-1 antibody is
a
mouse IgM and was detected
using
an
RITC-rabbit anti-mouse
IgM
(Axell)
for
1
h.
Paraffin sections were rehydrated
and
stained with
the
HNK-1 antibody as described above and elsewhere (Bronner-
Fraser, 1986). After antibody staining,
the
cell nuclei
in the
same sections were stained
by
placing them
in
0.2/tgml"
1
of
4-6-diamidino-2-phenylindole (DAPI) solution
in
0.1M
phos-
phate buffer
for
3
min.
Neural crest ablations
Chicken eggs were incubated until they reached stages
11-13
(according to the criteria
of
Hamburger and Hamilton, 1951).
A small hole
was
made
at one end of the egg and
1
ml
of
albumin
was
removed
to
lower
the
embryo away from
the
shell. A hole slightly larger than the blastoderm was cut
in the
shell overlying the embryo. India
Ink
(Pelikan Fount; diluted
1:4 in
Eagle's Minimum Essential Medium containing
15
%
horse serum
and 10%
11-day chick embryo extract)
was
injected underneath
the
blastoderm
to aid in
visualizing
the
embryo.
The
vitelline membrane
was
removed with
an
electrolytically sharpened tungsten needle.
In order
to
remove
the
presumptive neural crest cells,
the
dorsal portion
of the
neural tube
was
excised using glass
needles
and
Dumont
no. 5
forceps.
The
rostral limit
of the
ablation
was
typically three
to
four somites above
the
most
recently formed somite.
A
length
of
dorsal neural tube
corresponding
to six or
more somite lengths
was
removed.
Following
the
operation, embryos were sealed with adhesive
tape
and
returned
to the
incubator
for 24h
prior
to
fixation.
Embryos were prepared
for
cryostat sectioning
and
immuno-
cytochemistry
as
described above.
Analysis
of
cell density
Longitudinal paraffin sections
of
5
/an thickness were stained
T-cadherin
expressed in caudal somite
17
with HNK-1 antibody and DAPI as described above. The
numbers of somites were counted using the most recently
formed somite as a landmark. The sections were viewed
through a SIT camera onto a video screen. On randomly
selected sections, somite borders were traced and the cell
nuclei in a given somite were counted. The line demarcating
the rostral and caudal halves of the sclerotome was
determined by the distribution of neural crest cells, which
selectively migrate through the rostral half
only.
To determine
the number of sclerotomal cells, the number of
HNK-1-
positive cells was substracted from the total number of cells in
the rostral half of the sclerotome. A Student's two-sided Mest
was used to determine the statistical significance of differ-
ences in the number of cells per unit area.
Immunoblotting techniques
Somites were dissected from stage 16-17 chick embryos at
levels between the 8th rostral- and 6th caudal-most somites.
The tissue was homogenized immediately in cold
10 mM
Tris-HCl, pH7.6,
2
HIM
CaCl
2>
2% Nonidet P40, lmM
dithiothreiol,
1 mM
phenylmethylsulfonyl fluoride, 50/iM
leupeptin,
5/IM
pepstatin and 4ngml~' aprotinin. Proteins
were separated by SDS-PAGE (Laemmli, 1970) and trans-
ferred to polyvinylidene difluoride transfer membranes
(Immobilon P, Millipore) for several hours or overnight
(Towbin
et
al.
1979). Non-specific binding was blocked with
5%
nonfat dry milk in TBST
(10 mM
Tris-HCl, pH8.0,
150 mM
NaCl, 0.05% Tween 20) for lh. T-cadherin was
probed with rabbit anti-T-cadherin antiserum (1:500, Ranscht
and Dours-Zimmermann, 1991) followed by
125
I-goat anti-
rabbit immunoglobulin (lxlCrctsmin"
1
ml"
1
) diluted in
TBST. Each incubation was for one hour and followed by five
washes in TBST. Reacted blots were exposed for 1-5
h
to
Kodak XAR5-film with an intensifying screen.
Results
Neural crest cells begin to emigrate from the neural
tube shortly after neural tube closure. The majority of
neural crest cells move ventrally through the rostral half
of each somite (Fig. 1), first at rostral axial levels of the
embryo and subsequently at more caudal levels.
Therefore, several stages of neural crest cell migration
exist simultaneously within a single embryo. Neural
crest migration is well advanced in the rostral (older)
portion of the embryo, when it
is
just beginning in more
caudal (younger) regions. We have performed most of
our analyses on stage 16-18 chick embryos because
early and advanced stages of neural crest cell migration
exist simultaneously in the embryo at these times.
Anti-T-cadherin
antiserum
primarily
detects
a
9Ox26^M
r
protein in somites
Since T-cadherin is expressed in a variety of neuronal
and non-neuronal tissues (Ranscht and Dours-Zimmer-
mann, 1991), immunoblots were used to determine if
the antiserum specifically identifies T-cadherin in
somite tissue. Somites dissected from stage 16-17
chicken embryos were homogenized in the presence of
Ca
2+
and protease inhibitors, separated by SDS-
PAGE and transferred to polyvinylidene difluoride
membranes. After reaction with anti-T-cadherin anti-
serum and
125
I-goat anti-rabbit IgG, a single polypep-
tide of approximately
90X10
3
M
T
was detected (Fig. 2).
This reactivity compares well with results reported
elsewhere (Ranscht and Dours-Zimmerman, 1991) and
indicates that the immunoreactivity described below
reflects genuine T-cadherin.
Fig.
1. Schematic diagram
illustrating the early pathways of
trunk neural crest cell migration.
Neural crest cells emerge from
the neural tube (NT) and proceed
either ventrally (indicated by
large curved arrow) through the
somitic sclerotome (Scl) or
dorsolaterally (indicated by small
curved arrow) between the
ectoderm (Ec) and
dermomyotome (DM). The
ventrally migrating cells only
move through the rostral (R) half
of each sclerotome and are not
observed in the caudal (C) half
(Rickmann
et
al.
1985; Bronner-
Fraser, 1986). Neural crest cells
also avoid the region around the
notochord (No) (Pettway
et al.
1990) Ao=dorsal aorta.
18
B. Ranscht and M. Bronner-Fraser
-3
x10
200-
116-
97-
66-
Fig. 2. Reactivity of T-cadherin
antiserum on immunoblots. In
somites isolated from stage 16-17
chicken embryos, the anti-T-
cadherin antiserum used in this
study recognizes a single
polypeptide band of approximately
Appearance of
T-cadherin
in stage 16-18 chick
embryos
T-cadherin immunoreactivity within somite tissue was
observed in the caudal half of the sclerotome at all times
examined. A gradation of staining intensity was
observed along the rostrocaudal axis with most intense
immunoreactivity at rostral levels (Fig.3A,D). Using
the last-formed somites as reference, the caudal-most
region containing detectable T-cadherin immunoreac-
tivity was about three somites rostral to the last-formed
somite; T-cadherin appeared on a small population of
cells in the caudal portion of this somite (Fig. 3C). The
level of immunoreactivity increased with distance from
the last somite, with rostral portions of the embryo
expressing higher levels of T-cadherin than caudal ones
(Fig. 3A,D).
As the somites completed the epithelial-mesenchymal
transition to form the dermomyotome and sclerotome,
T-cadherin expression became restricted to the sclero-
tome. In newly formed sclerotomes, T-cadherin immu-
noreactivity appeared as a diffuse signal. Although the
immunoreactivity was most prominent in the caudal
half of the somite, there was no sharp discontinuity in
staining intensity distinguishing the rostral from caudal
halves of the sclerotome. Rather, the transition
between the caudal and rostral halves was diffuse and
gradual. Approximately six somites rostral to the last
formed somite, the level of immunoreactivity in the
caudal somite region intensified markedly. In addition
to the prominent immunoreactivity in the caudal halfof
the somite, a few faint fibrils of T-cadherin immuno-
reactive material appeared to extend into rostral
portions of the sclerotome.
Eleven to twelve somites rostral to the last formed
somite, both the morphology of the somites and the
T-cadherin immunoreactivity underwent an apparent
transition. T-cadherin expression appeared more dis-
tinct, with a sharp discontinuity in immunostaining
separating the caudal from the rostral halves of the
sclerotome. At this stage and beyond, T-cadherin was
clearly associated with the surfaces of caudal sclero-
tomal cells (Fig. 4), and absent from the rostral
sclerotome cells. Morphologically, the somites ap-
peared more compact and shorter in rostral-caudal
extent than somites in less mature regions of the
embryo.
In transverse sections at the level of the wingbud,
T-cadherin immunoreactivity in the sclerotome ap-
peared on the surface of
all
cells in the caudal half of the
sclerotome including the dorsal portion of the sclero-
tome, the region between the dermomyotome and
neural tube, and the ventral portion of the sclerotome
surrounding the notochord (Fig. 5A). In contrast,
immunoreactivity was absent from all regions of the
rostral half of the sclerotome. In more caudal (younger)
regions of the embryo, T-cadherin immunoreactivity
was observed in the dorsal sclerotome, but appeared to
be absent from the ventral sclerotome surrounding the
notochord (Fig. 5B). The dermomyotome and subepi-
dermal region appeared to lack T-cadherin immuno-
reactivity at all times examined. Migrating neural crest
cells were not seen to express T-cadherin, though motor
axons that also move through the rostral half of the
sclerotome, expressed T-cadherin at all stages exam-
ined.
T-cadherin
expression
in stage 16-18 embryos
inversely
correlates
with neural
crest
cell migration
The first neural crest cells to emigrate from the neural
tube in stage 16
—18
chicken embryos can be observed
three somite lengths rostral to the last-formed somite
(Bronner-Fraser, 1986). Although HNK-1-positive
neural crest cells emerge in an unsegmented pattern
along the entire dorsal surface of the neural tube, they
only enter the rostral half of each somite. The first
neural crest cells enter the somites at the time of
sclerotome formation. In embryos double-labelled with
anti-T-cadherin and FfNK-1 antibodies, we observed
neural crest cells entering the rostralmost portion of the
somite slightly after or concomitant with the appear-
ance of T-cadherin in the caudalmost portion of the
same somite.
The number of neural crest cells progressively
increased with distance from the last somite. Relatively
few neural crest cells were detected in the ten
caudalmost somites. Those were widely dispersed
throughout the rostral half of each sclerotome and had
no clear orientation. The distribution of the HNK-1
neural crest cells was complementary to the diffuse
staining for T-cadherin in the caudal halves of the same
somites (Fig. 3B).
In more mature regions of the embryo (11 or more
segments above the most recently formed somite), the
somites appeared more compact. Larger numbers of
neural crest cells with a clear orientation in the
Fig.
3. Fluorescence photomicrographs illustrating the distribution of T-cadherin (A, C, and
D) and HNK-1 antigen (B) in longitudinal sections through stage 17 embryos.
(A,B) T-cadherin is present in the caudal half of each sclerotome whereas neural crest cells,
recognized by the HNK-1 antibody are present in the rostral
half.
The T-cadherin
immunoreactivity is graded along the rostrocaudal axis with more intense staining in more
rostral regions. (C) High magnification view of the third somite above the most recently
formed somite of the embryo picture in A,B- T-cadherin immunoreactivity appears in a few
cells in the caudalmost region of the somite, with more diffuse staining in the caudal
sclerotome; neural crest cells in this sections (not shown) are situated between the neural
tube and somite and about to enter the rostral portion of the somite. This somite represents
the caudal limit .of the T-cadherin staining. (D) Another stage 17 embryo illustrating the
gradation in T-cadherin staining from more rostral to more caudal levels. Bar=209, 209, 20,
and
80
/an in A,D, respectively.
Fig.
4. Fluorescence photomicrographs of sagittal sections through the rostral somites of a
stage 17 embryo. (A) Double exposure micrograph of a section illustrating the distribution
of neural crest cells (red) and T-cadherin (green), which are inversely correlated.
(B,C) Another section snowing the distribution of neural CTest cells (B) and T-cadherin (C).
The neural crest cells are restricted to the rostral half of the sclerotome whereas T-cadherin
is in the caudal half of the sclerotome, where it appears associated with the somitic cell
surface. R=rostral, C=caudal, DM=dermomyotome. Bar=40/an.
T-cadherin expressed in caudal somite
19
mediolateral direction were migrating through the
rostral half of each sclerotome. At these more mature
axial levels, the expression of T-cadherin was more
pronounced than in more caudal regions of the embryo.
The boundary between the T-cadherin-positive caudal
halves and T-cadherin-negative rostral halves of the
sclerotomes was sharp, with T-cadherin expression
alternating with the HNK-1 -positive neural crest cells
(Fig. 4).
T-cadherin expression in stage 19 embryos
By stage 19, neural crest cells along the ventral pathway
Fig. 5. Fluorescence photomicrographs of transverse
sections through the rostral (A) and caudal (B) portions of
stage 18 embryos after staining with T-cadherin. (A) A
glancing section with the left-hand side through the rostral
(R) sclerotome and the right-hand side through the caudal
(C) sclerotome. T-cadherin immunoreactivity was
prominent on the motoraxons (indicated by curved arrow).
Although T-cadherin immunoreactivity was absent from the
rostral sclerotome, it was noted throughout the caudal
sclerotome, including the region around the notochord (N).
(B) In more caudal regions of the embryo, T-cadherin
immunore activity appeared
fibril
l ar within the caudal
sclerotome and was faint or absent in the neural tube.
Immunoreactivity was absent from the notochordal region.
Intense staining was observed on primordial germ cells
(indicated by straight arrows). Bar=47^m.
are in their final stages of migration and are beginning
to aggregate to form dorsal root and sympathetic
ganglia. At this time (Fig. 6), T-cadherin immunoreac-
tivity remained prominent in the caudal half of the
sclerotome, but now was also observed throughout the
myotome, though it was absent from the dermomyo-
tome at earlier stages. It contrast to the sclerotome,
staining of the myotome was non-segmented.
T-cadherin was detected on motor axons that, like
neural crest cells, move through the rostral half of each
sclerotome.
Cell density in the somites
One possible explanation for the absence of neural crest
cells from the caudal half of the sclerotome is that close
cell packing in this region may mechanically exclude
neural crest cells. To investigate if T-cadherin ex-
pression correlates with a possible compaction of the
caudal-half sclerotomal cells, we compared the length
of the somites and the density of sclerotomal cells in the
rostral
versus
caudal halves of the sclerotome. The
number of nuclei in a
1200
^m
2
area was determined in
randomly chosen sections through the rostral and
caudal halves of the sclerotome at 5, 7, 9, 13, and 15
somites above the last-formed somite in stage 16-17
embryos. Nuclei of HNK-1-positive neural crest cells
were substracted prior to quantitation. The presence or
absence of HNK-1 immunoreactivity also distinguished
the rostral half (neural crest-containing) from the
caudal half (neural crest-free) of the sclerotome. The
cell density in the caudal half of the sclerotome was
43-67%
higher than that in the rostral half at all somite
levels examined (P=s0.001; Table 1). Therefore, the cell
density in the caudal half of the sclerotome increases
Fig. 6. Fluorescent photomicrographs of longitudinal
sections through a stage 19 embryo. (A) At the level of the
ventral neural tube, neural crest-derived Schwann cells and
motoraxons stain with the HNK-1 antibody. (B) In the
same section, T-cadherin immunoreactivity is observed on
the motoraxons in the rostral (R) half of the sclerotome as
well as in the caudal (C) half sclerotomal cells, myotomal
(M) cells and cells in the ventral neural tube (NT).
Fig. 7. Fluorescent photomicrographs of two embryos fixed
at stage 17 from which the dorsal neural tube was removed
by surgical ablation at stage 11-12. Longitudinal sections are
illustrated at the level of the neural tube (NT). Even in the
absence of neural crest cells assessed by the lack of HNK-1
immunoreactivity in the rostral half of the sclerotome,
T-cadherin immunoreactivity is observed in the caudal half of
the sclerotome (B), where it appears on schedule. R=rostral;
C=caudal; DM=dermomyotome. Bar=40/an.